It has become evident that naturally occurring proteins fail to exhibit their inherent biological activity when their sugar chains are removed (A. Kibata, Tanpakushitsu Kakusan Koso 36, 775-788 (1991)). This suggests that sugar chains play an important role in developing biological activity. However, because the correlation between sugar chain structure and biological activity is not always apparent, the development of techniques allowing flexible modification and control of the structures (the types of sugars, the linked positions, chain lengths, etc.) of sugar chains attached to proteins is needed.
Glycoprotein sugar chains are largely classified as Asn-linked types, mucin types, O-GlcNAc types, GPI anchored types and proteoglycan types (M. Takeuchi, Glycobiology Series 5, Glycotechnology; edited by A. Kibata, S. Hakomori, K. Nagai, Kodansha Scientific, 191-208 (1994)), each of which have unique routes of biosynthesis and carry out different physiological functions. The biosynthesis pathway for Asn-linked sugar chains has been widely studied and analyzed in detail.
Biosynthesis of Asn-linked sugar chains begins with synthesis of a precursor consisting of N-acetylglucosamine, mannose and glucose on a lipid carrier intermediate, and its transfer to a specific sequence (Asn-X-Ser or Thr) of the glycoprotein in the endoplasmic reticulum (ER). It then undergoes processing (cleavage of the glucose residue and a specific mannose residue) to synthesize an M8 high mannose-type sugar chain composed of 8 mannose residues and 2 N-acetylglucosamine residues (Man8GlcNAc2). The protein including the high mannose-type sugar chain is transported to the Golgi apparatus where it undergoes various modifications, and these modifications at the Golgi apparatus differ significantly between yeast and mammals (Kukuruzinska et al., Ann. Rev. Biochem., 56, 915-944 (1987)).
In mammalian cells, one of three different pathways are taken, depending on the type of protein undergoing the sugar chain modification. The three pathways are cases 1) where the core sugar chain is not altered, 2) where the N-acetylglucosamine-1-phosphate moiety (GlcNAc-1-P) of UDP-N-acetylglucosamine (UDP-GlcNAc) is added at the 6-position of Man of the core sugar chain producing Man-6-P-1-GlcNAc, after which only the GlcNAc moiety is removed, for conversion to a glycoprotein having an acidic sugar chain, and 3) where five molecules of Man are removed in order from the core sugar chain, leaving Man3GlcNAc2 onto which almost simultaneously GlcNAc, galactose (Gal) and N-acetylneuraminic acid (also known as sialic acid (NeuNAc)) are added in order, resulting in a mixture of diverse hybrid and complex sugar chains [R. Kornfeld and S. Kornfeld, Ann. Rev. Biochem., Vol. 54, p.631-664 (1985)] (FIG. 1). Thus, it has been found that mammalian sugar chains have a variety of structures which are closely related to the functions of glycoproteins.
On the other hand, it has been found that yeast produce mannan-type sugar chains, or “outer chains”, having several to a hundred mannose residues on the above-mentioned core sugar chain (Man8GlcNAc2), while acidic sugar chains are also produced having mannose-1-phosphate added to the core sugar chain moiety and outer chain moiety (see FIG. 2). This modification differs from that of animal cells, and it has been reported that in yeast it does not function as a sorting signal for localization of glycoproteins to vacuoles (organelles corresponding to lysosomes in animal cells). The physiological function of phosphorylated sugar chains in yeast has therefore remained a mystery [Kukuruzinska et al, Ann. Rev. Biochem., Vol.56, p915-944 (1987)].
As shown in FIG. 2, the phosphorylation sites of mannose phosphate-containing sugar chains in yeast are sometimes added to the α-1,3 branch side and α-1,6 branch side of the Man8GlcNAc2 core sugar chain synthesized in the ER, and are sometimes added to the α-1,2 branches abundantly present on mannose outer chains synthesized in the Golgi apparatus, or to the non-reducing ends of mannose outer chains [Herscovics and P. Orlean, FASEB J., Vol. 7, p540-550 (1993)].
Biosynthesis of outer chains in Saccharomyces yeast is believed to occur along the pathway shown in FIG. 2 [Ballou et al., Proc. Natl. Acad. Sci. USA, Vol.87, p3368 (1990)].
Specifically, an elongation initiating reaction occurs wherein mannose is added to the M8 high mannose-type sugar chains at the α-1,6 linkages (FIG. 2, Reactions I, B). It has been shown that the enzyme responsible for this reaction is a protein encoded by the OCH1 gene (Nakayama et al., EMBO J., 11, 2511-2519 (1992)). Also, a reaction of successive elongation of mannose by α-1,6 linkages (FIG. 2: II) forms poly α-1,6-linked mannose as the skeletons of the outer chains (FIG. 2: E). The α-1,6-linked mannose has α-1,2-linked mannose branches (FIG. 2: C, F, H), and α-1,3-linked mannose is often added to the ends of the branching α-1,2-linked mannose (FIG. 2: D, G, H, I). The addition of these α-1,3-linked mannoses is performed by MNN1 gene product (Nakanishi-Shindo et al., J. Biol. Chem., 268, 26338-26345 (1993)). It has also become evident that some acidic sugar chains are produced having mannose-1-phosphate added to the high mannose-type sugar chain moieties (FIG. 2: *) and outer chain moieties. This reaction has been shown to depend on a protein encoded by the MNN6 gene (Wang et al., J. Biol. Chem., 272, 18117-18124 (1997)), while a gene (MNN4) has also been identified which codes for a protein which positively controls this transfer reaction (Odani et al., Glycobiology, 6, 805-810 (1996); Odani et al., FEBS letters, 420, 186-190 (1997)).
In most cases, outer chains result in heterogeneous protein products, both complicating protein purification and lowering specific activity (Bekkers et al., Biochim. Biophys. Acta, 1089, 345-351 (1991)). Moreover, because of the vast differences in sugar chain structures, glycoproteins produced in yeast do not exhibit the same biological activity as those from mammals, and are strongly immunogenic in mammals. For example, it is known that the α-1,3 mannoside linkages produced by the MNN1 gene in S. cerevisiae have strong immunogenicity (Ballou, C. E., Methods Enzymol., 185, 440-470 (1990)). It has been also reported that yeast inherently possess mannose-6-phosphate (Man-6-P) in the form of mannose-6-phosphate-α-1-mannose (Man-6-P-1-Man), which do not bind to Man-6-P receptors (Kukuruzinska et al., Annu. Rev. Biochem., 56, 915-944 (1987); Faust and Kornfeld, J. Biol. Chem., 264, 479-488 (1989); Tong et al., J. Biol. Chem., 264, 7962-7969 (1989)). Thus, yeast are considered unsuitable as hosts for production of useful mammalian glycoproteins. It has been a desire in both academia and industry to develop yeast that can produce glycoproteins with sugar chains having mammalian-equivalent biological activity, i.e., mammalian-type sugar chains. The present inventors have previously succeeded in creating mutants lacking outer chains and yeast with mammalian-type sugar chains (Japanese Patent Application No. 11-233215).
As mentioned above, yeast produce acidic sugar chains having mannose-1-phosphate added to the core sugar chain moiety and outer chain moiety by the action of the MNN4 gene and MNN6 gene. It has been demonstrated that sugar chains of glycoproteins produced by mutants which produce the core sugar chain and are deficient in the genes for the outer chain synthetic enzymes, include both neutral sugar chains (FIG. 3: Structural Formula I) and acidic sugar chains (FIG. 3: Structural Formulas II to IV) (Proceedings of the 12th Biotechnology Symposium, p.153-157, Oct. 14, 2004, Biotechnology Developmental Technology Research Society).
Such acidic sugar chains have a structure not found in mammalian sugar chains. Specifically, in mammalian cells it is not mannose-1-phosphate but rather N-acetylglucosamine-1-phosphate which is added, after which the N-acetylglucosamine moiety alone is removed to produce the final acidic sugar chain marked as “*” in FIG. 1. As will be further explained below and is taught in Methods in Enzymology, [Vol.185, p.440-470 (1990)], this sugar chain serves as a lysosome transport signal in mammalian cells.
Lysosomes are intracellular organelles containing numerous acidic hydrolases which decompose substances taken into the lysosomes both from within and without the cell. Most of the enzyme groups localized in human lysosomes, once biosynthesized and transported to the Golgi apparatus, undergo addition of phosphate groups at the 6-positions of mannose residues at the non-reducing ends of high mannose-type sugar chains, being thereby converted to acidic sugar chain-bearing glycoproteins, and the phosphate groups serve as lysosomal enzyme-specific recognition markers. They are distinguished from other proteins through binding with high affinity mannose-6-phosphate receptors (MPRs), and are carried into prelysosomes where they dissociate from the MPRs in the acidic environment and are then transported to lysosomes (von Figura and Hasilik, Annu. Rev. Biochem., 54, 167-193 (1984)). The binding with mannose-6-phosphate receptors (MPRs) requires that each sugar chain contain one or more mannose-6-phosphate molecules. This lysosomal enzyme-specific phosphate group addition is accomplished by two separate enzyme reactions. W. Canfield et al. have succeeded in cloning the genes for two enzymes (GlcNAc-phosphotransferase, GlcNAc-phosphodiester-GlcNAc′ase) involved in mannose-6-phosphate synthesis (Abstract of the XV International Symposium on Glycoconjugates. Glycoconjugate Journal Vol. 16 No. 4/5 S41 (1999)).
Genetic defects in these lysosomal enzymes, in enzymes involved in the phosphate-addition reaction or in factors contributing to activation or stabilization of the lysosomal enzymes causes a group of diseases characterized by blockage of the enzyme reactions and accumulation of intracellular substrates, such diseases being referred to collectively as “lysosomal disease” (Leroy and DeMars, Science, 157, 804-806 (1967)). Over 30 different types of lysosomal disease are known in humans and together they constitute an important disease group in pediatric and internal medicine. Strategies for developing basic treatments for such diseases have included bone marrow transplantation and gene therapy. Enzyme supplementation therapy using lysosomal enzymes has also been attempted, but poor uptake by target organs has been a major obstacle and at the current time the only satisfactory results have been seen with enzyme supplementation for Gaucher disease.
Gaucher disease results from a mutation in the gene for glucocerebrosidase, a glucosylceramide-degrading lysosomal enzyme, leading to accumulation of its substrate glucosylceramide mainly in bone marrow macrophage-derived cells, and manifested as notable hepatosplenomegaly as well as hematopoietic dysfunction including anemia and hemorrhage. As mentioned above, treatment methods for this disease include enzyme supplementation therapy, which has produced favorable treatment results, but such therapy must be continued for life and the enzyme preparations are extremely expensive. Glucocerebrosidase preparations are produced by modifying the ends of the sugar chains of human recombinant glucocerebrosidase expressed by CHO cells, to a form with the mannose exposed. Since the morbid cells in this disease are primarily macrophages, glucocerebrosidase is presumably transported to lysosomes after being taken up into the cells via mannose receptors on macrophages. Glucocerebrosidase is known to be transported into lysosomes regardless of whether it has mannose-6-phosphate on its sugar chains. It is therefore conjectured that the enzyme is transported to lysosomes by a mannose-6-phosphate receptor (MPR) non-dependent transport mechanism.
However, the deficient enzymes in most other lysosomal diseases are transported to lysosomes by mannose-6-phosphate receptor-mediated systems, and therefore the lysosomal enzymes used for enzyme supplementation therapy must having mannose-6-phosphate-containing sugar chains as the lysosome migration signals necessary for binding with mannose-6-phosphate receptors (MPRs). Addition of mannose-6-phosphate to these lysosomal enzyme chains is therefore a key strategy.
Currently, the reported methods for obtaining lysosomal enzymes include methods of purification from placenta, production methods utilizing cultured cells such as fibroblasts or melanoma cells, recombinant methods using cultured cells such as insect cells or Chinese hamster ovary (CHO) cells, and methods of obtaining the enzymes from transgenic rabbit milk. However, these methods are associated with the disadvantages of 1) low content of lysosomal enzymes with phosphate-added sugar chains and therefore poor uptake efficiency into lysosomes, and 2) low productivity/high culturing cost. Disadvantage 1) therefore requires high-dose administration, while disadvantage 2) leads to high treatment costs. Moreover, production by recombinant methods using yeast cells has not yet been achieved. Thus, enzymes having mannose-6-phosphate on the sugar chains and having high uptake activity into lysosomes have been a desired goal.
One of the lysosomal disease known as Fabry disease is an X-chromosomal genetic disease characterized by reduced α-galactosidase activity and accumulation of its in vivo substrate globotriosylceramide in the body. Fabry disease patients in the classic type of the disease typically suffer extremity pain, cutaneous hemangioma and impaired sweating beginning from youth or adolescence, and exhibit nephropathy or cardiovascular and cerebrovascular disorders with increasing age. In recent years, a relatively mild Fabry disease “subtype” has been identified which is marked by cardiomyopathy in late middle age or thereafter, and it has been reported that such patients may be hidden among patient groups falsely diagnosed with cardiomyopathy.
α-Galactosidase differs from the aforementioned glucocerebrosidase in that it is transported to lysosomes by a mannose-6-phosphate receptor-mediated system. Consequently, it must have mannose-6-phosphate-containing sugar chains in order to be taken up efficiently by the target cells. However, the technology has not existed for mass production of high-purity α-galactosidase with mannose-6-phosphate-containing sugar chains, suitable for use in therapy. For example, the proportion of mannose-6-phosphate sugar chains is thought to be about 20% in fibroblast-derived glycoprotein (α-galactosidase), which has shown superiority as an enzyme supplementation infusion in 9 out of 10 human patients (Pro. Natl. Acad. Sci. USA, 97:365-370 (2000)).
Since the structures of the sugar chains added to proteins differ in yeast and mammalian cells, useful human or other mammalian glycoproteins produced in yeast by genetic engineering methods do not exhibit identical activity as those derived from mammals, or they may have different antigenicities due to the different sugar chains. It has been difficult to produce mammalian glycoproteins in yeast for this reason. Furthermore, while useful phosphate-containing acidic sugar chains having the identical sugar chain structures as are added in human and other mammalian cells would be of benefit in functioning as labeling markers for transport of the glycoproteins to lysosomes in human or other mammalian cells, it is currently difficult to supply such acidic sugar chain-having glycoproteins in uniform, large amounts. The development of such technology has therefore been greatly desired.
The present inventors have previously proposed a method composing using a sugar chain synthesis mutant (ΔOCH1 mnn1) to produce a glycoprotein, allowing the MNN6 gene product to act thereon either in vivo or in vitro to obtain mannose-1-phosphate-added acidic sugar chains, and carrying out acid treatment to obtain mammalian-like sugar chains which are effective as lysosome transport signals (Japanese Unexamined Patent Publication HEI No. 9-135689). However, due to the extreme denaturing conditions used for this method (0.01 N hydrochloric acid, 100° C., 30 minutes), virtually all of the glycoprotein becomes denatured. It has therefore been unsatisfactory as a method for obtaining glycoproteins with physiological activity.